The goal of this project is to 1) understand the role of mitochondria in ischemia-reperfusion injury and cardioprotection and 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection. Isolated mitochondria from mice deficient in cyclophilin D (CypD-/-) are less sensitive to Ca2+-induced opening of the mitochondrial permeability transition (MPT) in vitro. Thus, the lack of CypD enables heart mitochondria to take up more Ca2+ before undergoing the MPT. We hypothesize that the MPT serves as a Ca2+-safety valve that can open to release excess Ca2+, but not necessarily result in death. If the MPT is blocked in CypD-/- mice, we hypothesize that matrix Ca2+ (Ca2+m) would be higher in CypD-/- mice compared to WT and this would activate Ca2+-sensitive NADH dehydrogenases (e.g., pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH)), which would in turn, alter oxidative metabolism and increase oxygen consumption. Consistent with this, we found altered expression levels of PDH E1 subunit and the alpha-KGDH E2 subunit in CypD-/- hearts using 2D DIGE proteomics. To evaluate differences in metabolism, we perfused hearts with 13C-glucose and 13C-palmitate and looked at their contribution to the acetyl-CoA pool by measuring label incorporation into the C4 of glutamate. The 13C-labeled glucose or palmitate enters the Krebs cycle and labels the alpha-KG pool that is in equilibrium with glutamate, which is usually present at higher levels. The ratio of glucose to palmitate metabolism in CypD-/- hearts was 1.5-fold higher than in WT, which would suggest increased PDH activity. 13C-labeling into succinate compared to glutamate was also increased significantly in CypD-/- hearts, and this result would be consistent with increased activity of alpha-KGDH relative to other competing reactions. We measured alpha-KGDH activity to evaluate whether Krebs cycle flux upstream of succinate was elevated in CypD-/- hearts and found a 1.4 fold increase in alpha-KGDH activity. Therefore, these results demonstrate that the loss of a MPT component, CypD, results in physiological flux changes in the Krebs cycle and oxidative metabolism that are consistent with increased Ca2+m. We also performed studies to examine the role of nitric oxide in cardioprotection. Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion;(2) IPC;or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca2+ channel alpha1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca2+-ATPase, alpha-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase alpha1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart. We also examined mechanisms by which inhibition of glycogen synthase kinase (GSK) mediates cardioprotection. Inhibition of GSK-3 reduces ischemia-reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to determine the molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSK inhibitors prior to ischemia, significantly improved recovery of function. To assess the effect of GSK inhibitors on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSK inhibitors and treated with uncoupler or cyanide, or were made anoxic. We found that GSK inhibition slowed ATP consumption under these conditions, which could be due to inhibition of ATP entry into the mitochondria through VDAC and/or ANT or to inhibition of the F1F0 ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer but not the inner mitochondrial membrane, and we found that GSK inhibitors slow AMP production similar to their effect on ATP consumption. This suggests that GSK inhibitors are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not due to opening of the mitochondrial permeability transition pore. Since GSK is a kinase, we wanted to determine if protein phosphorylation might be involved. Therefore, we performed western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSK inhibitors. LC/MS analysis revealed one of these proteins to be VDAC2. Taken together, we found that GSK mediated signaling modulates transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest that GSK regulates VDAC and suggest that VDAC may be an important regulatory site in ischemia-reperfusion injury. Most of these studies showing that inhibition of GSK is protective, however, were performed in rats. We therefore did additional studies to determine whether GSK- inhibition mimicked the protective effects of PC in mice. Langendorff murine hearts were treated with a specific GSK-3 inhibitor AR-A014418 (GSK Inhib VIII) for 10 min or subjected to 4 cycles of 5-min ischemia/reperfusion (PC) prior to a 20-min global ischemia and 120-min reperfusion. PC and GSK had improved post-ischemic LVDP recovery compared to their time-matched controls (57.0 2.5 and 51.7 3.8% vs. 33.6 2.4% of their pre-ischemic LVDP). Consistent with improved functional recovery, infarct size in PC and GSK Inhib VIII hearts was decreased relative to control (11.9 1.6 and 19.2 3.5 vs. 34.8 1.9%). Lactate levels were measured in hearts snap-frozen after a 20-min ischemic period and found to be significantly lower in PC and GSK Inhib VIII-treated groups (6.69 0.73 and 8.30 0.29 moles lactate/g wet weight) with respect to control (10.34 0.67 moles lactate/g wet weight). We used a comparative proteomics analysis to look at mitochondrial protein expression/phosphorylation changes that were present only in PC and GSK Inhib VIII-treated, but not in control. Levels of ATP synthase e, cytochrome c oxidase subunit VIb, ATP synthase coupling factor 6, cytochrome c oxidase subunit 5A, and cytochrome b-c1 complex subunit were increased while cytochrome c was decreased in PC and GSK Inhib VIII. These changes were not the result of alterations in protein expression and but post-translational modifications. PC and GSK Inhib VIII hearts had reduced levels of phospho-Tyr cytochrome c as measured with Western blot.